TARGETED REPLACEMENT OF NORMAL AND MUTANT CFTR SEQUENCES IN HUMAN AIRWAY EPITHELIAL-CELLS USING DNA FRAGMENTS

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator (CFTR) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed prima ry normal airway epithelial cells. The endogenous genotype of these no rmal cell lines was modified following liposome or dendrimer transfect ion using DNA fragments with Delta F508 CFTR sequence (488 nt, complem entary single strands) designed to also contain a unique restriction e nzyme cleavage site (XhoI), Replacement at the appropriate genomic loc us by exogenous Delta F508 CFTR DNA and its expression as mRNA was dem onstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as XhoI digestion of the PCR products. These studies show that S FHR occurs in both transformed and non-transformed primary human airwa y epithelial cells and indicate that single base substitution (the sil ent mutation giving rise to the XhoI site) and deletion or insertion o f at least three consecutive bases can be achieved in both normal and CF epithelial cells, Furthermore, these studies reiterate the potentia l of SFHR as a strategy for a number of gene targeting applications, s uch as site-specific mutagenesis, development of transgenic animals, d evelopment of isogenic cell lines and for gene therapy.